Medicament containing an effector of the glutathione metabolism together with $g(a)-lipoic acid for treating diabetes mellitus

ABSTRACT

The invention relates to a medicament containing an effector of the glutathione metabolism together with α-lipoic acid for treating diabetes mellitus. This medicament enables disturbances of the thiol-disulfide status or those that occur, for example, in diabetes mellitus to be treated simultaneously, separately or in a temporally graduated manner.

[0001] This invention relates to the use of the combination of α-lipoicacid and effectors of the glutathione metabolism for the treatment ofdisruptions of the cellular thiol status and related illnesses.

[0002] The precise regulation of the thiol disulfide status representsone of the most important basic requirements of biological metabolisms.The central regulating element within this system is the tripeptideglutathione, which reaches high intracellular concentrations (up to 10mM) in reduced form. In addition to glutathione, protein groups carryingthiol groups intracellularly and particularly in cell-membrane-bondedform are additional important components of the thiol disulfide statusof each cell.

[0003] The metabolism of the breakup of disulfide and the formation ofthe thiol groups, which is regulated by various classes of enzymes, isessential for any normal cell function as a result of the variety of thebiological functions of thiol in, among other things, cell growth anddifferentiation processes including programmed cell death as well ascell protection and decontamination mechanisms overall. Disruptions inthis system and changes in the concentration of thiol lead to seriousdisruptions in cell function, which remain locally limited only inisolated cases, and generally have an adverse effect on the entireorganism.

[0004] The involvement of a disrupted thiol disulfide status in acuteand chronic diseases has been demonstrated in a number of experiments.

[0005] In neurodegenerative diseases such as Parkinson's Disease, forexample, significant changes in the thiol metabolism have beendemonstrated in certain nerve cells (Brain Res Rev 1997;25:335-358).There are clear indications that as a result of this metabolicdisruption, there is an increased death of nerve cells in thefunctionally impaired areas of the brain, the basal ganglia, which isprimarily responsible for the symptoms of the disease (Ann Neurol1994;36:348-355). Reduced glutathione levels and a reduced intracellularglutathione content have also been found in the context of angiopathiesand their consequences—arteriosclerosis and heart attack—in theendothelial cells that line the inner wall of the vessel. (Med Sci Res1998:26:105-106).

[0006] Pulmonary diseases that involve a transformation of the pulmonarytissue are regularly connected with a glutathione deficit in the tissue.In a pulmonary fibrosis of this type, the severity of the disease runsparallel to the thiol loss (Clin Chim Acta 1997;265:113-119). Seriousinflammatory pulmonary diseases, investigated using the example of acutedifficulty of breathing in adults, were accompanied by a dysregulationof the thiol metabolism of the participating inflammatory cells(granulocytes) (Chest 1996;109:163-166).

[0007] On the basis of the author's own tests, the immuno-competentdefensive cells of the bronchial system (alveolar macrophages) insmokers and in patients with chronic obstructive respiratory diseasesexhibit a serious cellular thiol deficit. The severity of the disruptionof the cellular thiol status is thereby directly correlated withrestrictions in lung function (Free Radic Biol Med 2000;29:1160-1165).

[0008] Extensive research into the significance of the glutathionemetabolism in viral infections has revealed both a poorer prognosis ofthiol-deficient cells based on a compromised cellular defense, as wellas an antiviral function of the glutathione that combats thereproduction of the virus (Proc Natl Acad Sci USA 1997;94:1967-1972).

[0009] The author's own tests have shown that the cellular thioldisulfide metabolism is seriously disrupted, particularly under theconditions of severely restricted kidney function and the resultingnecessary renal replacement therapy in the form of hemodialysis orperitoneal dialysis. This disruption results in, among other things, theextensive loss of normal cell functions, such as the phagocytosiscapability of peritoneal macrophages or the activation of lymphocytes.

[0010] The human cellular immune system, which consists of the whiteblood cells granulocytes, lymphocytes and monocytes, represents a systemthat reacts particularly sensitively to a disruption in the thiolmetabolism.

[0011] Minimal changes, particularly losses of cellular glutathione, cantrigger a cascade-like program for the self-destruction of the cells,the programmed cell death (apoptosis) (FASEB J 1998;12:479-486). In thiscase, the thiol disulfide metabolism acts as a central switchingmechanism of an intact immune system, without which the organism wouldnot be viable.

[0012] In recent years, moreover, there have been increasing referencesto a damaged thiol metabolism in chronic kidney diseases (Ren Fail1998;20:117-124), anemias (Br J Haematol 1995;91:811-819), prematurenewborns (Pediatr Pulmonol 1995;20:160-166), hearing loss caused bynoise (Brain Res 1998;784:82-90), inflammatory bowel diseases (Gut1998;42:485-492) and particularly in diabetes mellitus (Metabolism:Clinical and Experimental 1998; 47(8):993-997).

[0013] Studies in the context of diabetes mellitus and associatedmetabolic disruptions have demonstrated both a shift of the redox statusat the expense of reduced glutathione as well as an absolute reductionof the total glutathione pool (Free Radic Biol Med 1998;24:699-704). Inthe summary of the previous literature on the role of the disruption ofthe thiol disulfide status, it has been assumed that not only is therean accompanying SH deficit as a consequence of the primary disease,which is Type 1 or Type 2 diabetes, but that a dysregulation in thethiol metabolism is at least one factor that triggers the disease. Aconvincing example has been provided, among other things, by theverification of the radical-induced destruction of the pancreaticβ-cells (Diabet Med 2000;17:171-180).

[0014] It is also known that the disease is accompanied by a number ofimmunological disruptions. The primary factor that has been identifiedis an imbalance of the immunoregulator cytokine, which is related tofunctional disruptions of the lymphocytes and macrophages, with aresulting significant increase in the sensitivity of the patients toinfections (Horm Metab Res 1998;30:526-530).

[0015] The correction of a disrupted thiol metabolism thus acquiresfundamental importance as a basic therapy in the treatment of a numberof diseases of different geneses, particularly, however, under theconditions of diabetes mellitus.

[0016] α-lipoic acid has been used relatively successfully in the formof a neuro-protective substance for the treatment of neuro-toxicallycaused paresthesia in the context of diabetic polyneuropathy(Diabetologica 1995;38:1425-1433, Diabetes Res Clin Pract 1995;29:19-26,Diab Care 1999;22:1296-1301, Drug Metab Rev 1997;29:1025-1054, DE 43 43592 C2). DE 44 47 599 C2 and EP 0 530 446 B1 also describe the use ofα-lipoic acid in additional neuronal disruptions, including tinnitus andapoplectiform deafness.

[0017] In this case, the cytoprotective mechanism of action depends onthe influence of the sugar-dependent protein modification (proteinglycolysis), on a reduction of neurotoxic ketogenesis, and finally onthe anti-oxidation function of the α-lipoic acid and its metabolites(Free Radic Biol Med 1995;19:227-250).

[0018] This cell protection function has been investigated particularlyfrom the point of view of the prevention of the oxidative transformationof essentially unsaturated fatty acids. Such an inhibition of the lipidperoxidation, in addition to the use of the α-lipoic acid as aneuro-protection agent, represents the basis for an application as amedicament to protect the liver in the treatment of variousintoxications and liver diseases (Biochemistry 1998;37:1357-1364).

[0019] It has also been shown that α-lipoic acid inhibits thereproduction of the HIV virus in different stages of its growth and maythus counteract a progression of the AIDS disease. The results of theselaboratory tests can be applied to clinical studies only to a limitedextent, however (FEBS-Lett 1996;394:9-13). The same is true for thedetection of an anti-inflammatory function of the substance for theinsulin-producing islet cells of the pancreas (Agents Actions1993;38:60-65).

[0020] EP 0 812 590 A2 and EP 0 427 247 B1 disclose the use of α-lipoicacid as a cyto-protective, as an analgesic and as a medicament for thetreatment of inflammatory diseases.

[0021] The anti-oxidative properties of α-lipoic acid are based on itsability to form chelates with metal ions and to eliminate radicalsdirectly, as well as on its function as a strong reducing agent. Toperform this reaction on an intracellular level, α-lipoic acid, even inreduced form, must be present in the form of dihydrolipoic acid. Thetransition from (disulfide) α-lipoic acid by means of reduction into thedithiol form of dihydrolipoic acid for its part consumes reducingequivalents, whereby this process is catalyzed by, among other things,the enzyme glutathione reductase (Gen Pharmacol 1997;29:315-331). Thisprocess is apparently the cause of the unsatisfactory action of thesubstance in terms of thiol restitution.

[0022] Ambroxol, i.e.trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexane hydrochloride, isadministered in various forms as an expectorant in the treatment ofpulmonary and bronchial diseases (WO 96 33704, GB 2239242, WO 01 05378).Its use in the treatment of hyperuricemia is also known from DE 35 30761. The action of ambroxol as a mucolytic is based both on astimulation of surfactant production by the bronchial cells andparticularly on its ability to eliminate free radicals (Respir Med1998;92:609-23). The anti-oxidative activity of the substance based onthese properties was demonstrated primarily on pulmonary cells(Pharmacol 1999;59:135-141), but also in the context of inflammatorymechanisms (Inflamm Res 1999;48:86-93). It is also known that throughthe use of ambroxol in high doses, enzymes that regulate the glutathionemetabolism can be influenced directly and peroxidative processes can beinhibited in vitro (Arch Vet Pol 1992;32:57-66).

[0023] Inhibitors of the angiotensin-converting enzymes(Angiotensin-Converting Enzyme Inhibitors, ACE inhibitors) have beenused with great success in the treatment of a wide range ofcardiovascular diseases. The mechanism of the effect of reducing bloodpressure is based on the inhibition of the conversion of angiotensin Iinto angiotensin II. ACE inhibitors have also been described aseffectors of the glutathione metabolism. In addition to research ineffects of this type in the treatment of cardiovascular and vasculardiseases (J Cardiovasc Pharmacol 2000;36:503-509), general regulationprinciples have been investigated (Clin Nephrol 1997;47:243-247). Theeffects under investigation in that case were the effects of ACEinhibitors that carry SH groups such as Captopril(-[(2S)-3-mercapto-2-methylpropionyl]-L-prolin), of SH-free ACEinhibitors such as enalapril(1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-prolin). Theformer react directly as radical interceptors antioxidatively, whileSH-free ACE inhibitors are essentially incapable of doing that. Acapability common to both groups is their influence on the glutathioneredox cycle by means of the regulation of glutathione reductase andglutathione peroxidase, as well as of superoxide dismutase (Am J.Physiol Regulatory Integrative Comp. Physiol. 2000;278:572-577).

[0024] The object of the present invention was therefore to makeavailable novel medicaments containing thiol-reactive substances for theimproved stabilization of an impaired thiol disulfide status in thetreatment of diabetes mellitus and for the restitution of the functionallosses caused by said disease.

[0025] The invention teaches that this object can be achieved by themedicament according to the invention and having the features recited inclaim 1. Claims 13 to 15 recite the use of the actives for themanufacture of a medicament. The additional dependent claims each reciteadvantageous developments of the invention.

[0026] According to the invention, effectors of the glutathionemetabolism are used in combination with α-lipoic acid, its salts and/orits prodrugs.

[0027] The invention teaches that as a result of the application of thecombination of α-lipoic acid and an effector of the glutathionemetabolism used according to the invention, there is a normalization ofthe primary reduced thiol status of immune cells. Not only does thethiol-stabilizing action of the combinations regularly exceed that ofthe use of α-lipoic acid or of the respective effectors individually,but super-additive effects have also been found. The restitution of thethiol status thereby comprises both intra-cellular thiols as well asmembrane-bonded SH groups and is thus an expression of a complexbiological regulation. This phenomenon is based on the fact that theeffectors of the glutathione metabolism on the one hand eliminateintermediary free radicals that occur, and on the other hand increasethe availability of reducing equivalents for the transformation of theα-lipoic acid from disulfide into reduced form, and thus enhance thesynthesis-inducing effect of the α-lipoic acid on the thiol disulfidestatus.

[0028] It also became clear that an a thiol-increasing effect of thecombination of effectors of the glutathione metabolism and α-lipoic acidoccurred only in primarily thiol-deficient immune cells. Healthy immunecells, which do not have any alteration of the thiol disulfide status,did not react with a further increase of the SH concentration.

[0029] The restitution of the thiol status of the immune cells wasaccompanied by a normalization of functional parameters. This phenomenonrelated in particular to the immuno-modulatory effects in the context ofthe activation of T-lymphocytes.

[0030] It has also been demonstrated that the combinations usedaccording to the invention stabilize the thiol disulfide status ofadditional immune cells such as the peritoneal macrophages of patientswho required dialysis. The peritoneal macrophages from high-glucoseperitoneal dialysis fluids, prior to treatment with α-lipoicacid/ambroxol or α-lipoic acid/ACE inhibitors, in addition to adeficient thiol status, have an almost complete loss of theirphagocytosis function, as well as a serious disruption ofdifferentiation and cytokine synthesis, which have been described ascauses of the high infection rates in these patients. These functionallosses were eliminated by the addition of the combinations according tothe invention.

[0031] This medicament is particularly appropriate for the treatment ofdiabetes mellitus as well as other clinical pictures in which there is adisruption of the thiol disulfide status of the immune cells. Thetreatment can therefore be administered simultaneously, in separateformulations, or in a temporally graduated manner.

[0032] The combination preparations used according to the invention canbe administered in the conventional pharmacological forms ofadministration or in the form of an instillate, as well asprophylactically and therapeutically. The effective dose must thereby bedetermined on a case-by-case basis. The dose for applications in humanpatients is preferably between 30 and 1200 mg/d, with particularpreference given to doses between 200 and 600 mg/d.

[0033] In one variant, ambroxol having the general formula I

[0034] its salt and/or its prodrug are used as the effector of theglutathione metabolism. The dose of ambroxol for applications in humanmedicine is therefore preferably between 7.5 and 90 mg/d, withparticular preference given to doses between 60 and 75 mg/d.

[0035] In an additional variant, an inhibitor of theangiotensin-converting enzyme (ACE inhibitor) is used as the effector ofthe glutathione metabolism. In this case, the preferred dose forapplications in human medicine is between 0.2 and 20 mg/d.

[0036] The following compounds, for example, can be used as ACEinhibitors:

[0037] A) 1-[(2S)-3-mercapto-2-methylpropionyl]-L-prolin (Captopril)having the formula II

[0038] B) 1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-prolin(Enalapril having the formula III

[0039] C) (2S, 3aS,6aS)-1-{(S)-N-[(S)-1-ethoxycarbonyl-3-phenylpropyl}-alanyl}-octahydrocyclopenta[b]-pyrrol-2-carboxylic acid (Ramipril) having the formula IV

[0040] The medicament can thereby be administered orally and/orparenterally.

[0041] The medicament can also contain the conventional additives. Suchadditives include, for example, aqueous solvents, stabilizers,suspension agents, dispersion agents and wetting agents.

[0042] The medicament can be manufactured in any desired formulation. Byway of example, acceptable formulations include solutions, granulates,powder, emulsions, tablets and/or coated tablets.

[0043] According to the invention, an effector of the glutathionemetabolism together with α-lipoic acid, its salt and/or its prodrugs,are used for the manufacture of a medicament for the treatment of adisruption of the thiol disulfide status of immune cells in thetreatment of diabetes mellitus.

[0044] Likewise, an effector of the glutathione metabolism together withα-lipoic acid, its salt and/or its prodrugs can be used for themanufacture of a medicament for a treatment to modulate immunity,increase defenses or inhibit inflammation.

[0045] The components of the combination preparation can therefore be ina single formulation or in separate formulations.

[0046] The use of the combination of α-lipoic acid and effects of theglutathione metabolism according to the invention is described ingreater detail below with reference to examples and the accompanyingfigures.

EXAMPLE 1

[0047] Influence on the Cellular Thiol Status of Human Peripheral ImmuneCells.

[0048] Peripheral immune cells from healthy donors (n=9) were isolatedfrom the peripheral blood. The major fraction of the resulting totalpopulation of mononuclear cells regularly represents lymphocytes with arelative percentage of approximately 90%, depending on the donor. 10% ofthe mononuclear cells are represented by monocytes.

[0049] The mononuclear cells obtained were absorbed in special cellculture media and incubated in a gasifying incubation cabinet at 37° C.,a relative humidity of 98% and 5% relative air-CO₂ content. Themetabolism of the primarily inactive immune cells was activated by meansof mitogenic stimulation (0.5 μg/ml phytohemagglutinin). To test theinfluence of the combinations used according to the invention on thethiol status of thiol-deficient immune cells, these cells wereartificially thiol-depleted. This process was carried out using provenmethods by cultivation in thiol-deficient media (RPMI 1603). Comparativecultures using complete media (RPMI) 1640) were used to define the bestpossible normal value under the culture conditions.

[0050] The determination of the intercellular thiol content on thesingle-cell level was made using 5-chloromethylfluorescein diacetate(CMFDA) in flow cytofluorimetry.

[0051] Primarily non-fluorogenic CMFDA is thereby passively absorbed bythe cell. By means of the chlormethyl group, there is a bonding tocytoplasmic thiol groups. After the breakdown of the acetate groups bynon-specific cellular esterases, this complex, which is now cellmembrane bound, becomes fluorogenic at an excitation wavelengthλ_(ex)=490 nm with an emission wavelength λ_(em)=520 nm. The medianfluorescence intensity of the specimen (10,000 cells) is directlyproportional to the concentration of the intracellular thiol groups.

[0052] The expression of membrane-bound thiol groups was also determinedusing flow cytofluorimetry. In this case, chloromethyltetramethylrhodamine (CMTMR) was used as thiol conjugate under the conditions of ablocked membrane potential and an inhibited diffusion capacity of thecells. The fluorescence intensity of the fluorochrome molecules bound tothe cell membrane is in turn proportional to the number of thiol groupson the cell surface.

[0053]FIG. 1 illustrates the effect of the combination of α-lipoic acidand ambroxol (FIG. 1a) and α-lipoic acid and enalapril (FIG. 1b) on theintracellular thiol expression of lymphocytes. The following table showsthe results of the combination of α-lipoic acid and Captopril. The dataare presented as the ratio of the cellular fluorescence intensity to therespective calibration particles (beads) that were analyzedsimultaneously. The actives concentration of the respective combinationis identical to the concentrations of the individual components.Intracellular thiol expression [mfi_(Beads/Ratio)] α-lipoic α-lipoicLength of acid Captopril acid + culture [d] Control [50 μM] [10 μM]Captopril 0 2.88 ± 0.20 2.88 ± 0.20 2.88 ± 0.20 2.88 ± 0.20 1 2.31 ±0.20 2.81 ± 0.23 2.80 ± 0.21 2.89 ± 0.31 2 1.98 ± 0.16 2.76 ± 0.50 2.76± 0.22 2.92 ± 0.32 3 1.63 ± 0.15 2.63 ± 0.60 2.49 ± 0.26 2.88 ± 0.41 41.30 ± 0.16 2.41 ± 0.40 2.21 ± 0.36 2.91 ± 0.39 6 1.10 ± 0.13 2.23 ±0.50 1.83 ± 0.33 2.93 ± 0.35 8 0.95 ± 0.10 2.02 ± 0.30 1.02 ± 0.39 2.93± 0.41 10 0.81 ± 0.10 1.89 ± 0.30 0.91 ± 0.46 2.90 ± 0.45 12 0.69 ± 0.101.86 ± 0.68 0.76 ± 0.49 2.88 ± 0.49 14 0.65 ± 0.08 1.83 ± 0.60 0.75 ±0.56 2.86 ± 0.47

[0054] Peripheral immune cells were cultivated over a period of 4 daysunder standard (Control 1640) or thiol-deficient conditions (1603) forthe induction of a 10-20% thiol reduction. As illustrated in 1 a, theaddition of ambroxol in combination with α-lipoic acid beginning after48 hours of treatment resulted in a total compensation of theintracellular thiol deficit. Using the combination of α-lipoic acid andthe SH-free ACE inhibitor enalapril and the SH-carrying ACE inhibitorcaptopril, these effects were further increased quantitatively, and weredetectable after only 24 hours. It was not possible to achieve acomplete compensation of the thiol deficit using either α-lipoic acidalone or with the individual application of the effectors.

[0055] The results obtained with this experimental investigation of theinfluence of the combinations according to the invention on theexpression of cell membrane bound thiols are presented in FIG. 2 for thecombinations of α-lipoic acid and ambroxol (FIG. 2a) and α-lipoic acidand enalapril (FIG. 2b). The following table presents the results forthe combination of α-lipoic acid and captopril. Membrane-resistant thiolexpression [mfi_(Beads/Ratio)] α-lipoic α-lipoic Length of acidCaptopril acid + culture [d] Control [50 μM] [10 μM] captopril 0 2.37 ±0.45 2.37 ± 0.45 2.37 ± 0.45 2.37 ± 0.39 1 2.79 ± 0.50 2.65 ± 0.39 2.63± 0.39 2.38 ± 0.38 2 2.35 ± 0.45 2.43 ± 0.52 2.54 ± 0.41 2.42 ± 0.41 31.98 ± 0.43 2.31 ± 0.36 2.52 ± 0.38 2.49 ± 0.46 4 1.63 ± 0.43 2.26 ±0.20 2.50 ± 0.41 2.39 ± 0.52 6 1.10 ± 0.46 2.19 ± 0.13 2.46 ± 0.50 2.40± 0.50 8 0.98 ± 0.31 1.93 ± 0.20 2.01 ± 0.39 2.40 ± 0.53 10 0.96 ± 0.321.63 ± 0.16 1.68 ± 0.29 2.36 ± 0.52 12 0.95 ± 0.33 1.32 ± 0.21 1.02 ±0.51 2.38 ± 0.49 14 0.98 ± 0.33 1.34 ± 0.20 0.99 ± 0.46 2.36 ± 0.55

[0056] In the treatment using the combination of α-lipoic acid andambroxol, there was once again, beginning after 48 hours, a significantimprovement in the membrane-bound thiol expression. In this case, it wasparticularly noteworthy that the addition of the individual substancesdid not at any time have a significant effect. The addition of thecombination of α-lipoic acid and the respective ACE inhibitors resultedin a super-additive effect, both with enalapril and with captopril.

EXAMPLE 2

[0057] Influence on the Cellular Activation Status of Human PeripheralT-Lymphocytes

[0058] In the cultivation experiment described with reference to Example1, human T-lymphocytes were stimulated with 1.0 μg/mlphytohemagglutinin. In a culture time of 72 hours, specific markers ofthe cellular activation were quantitatively detected usingcytofluorimetery by means of monoclonal antibodies. The influence of thecombinations used according to the invention on the activation markersCD69 (early activation antigen), CD25 (intermediate activation antigen)and CD71 (late activation antigen) of T-lymphocytes was measured. FIG. 3shows the effect of the combination of α-lipoic acid and ambroxol (FIG.3a) and α-lipoic acid and enalapril (FIG. 3b) on the activation index ofT-lymphocytes. Compared with normal T-lymphocytes (activationindex=1.0), in thiol-deficient cells there is a clear reduction of theactivation, which demonstrates the disruption of cellular function.After the addition of α-lipoic acid, the well-known effect of a slightimprovement of the cellular activation occurs, which however in no casecaused any significant difference from the normal control group.Ambroxol does not have any influence on one of the three activationmarkers. The ACE inhibitor Enalapril is equivalent to α-lipoic acid onlyin the case of the effectuation of the CD25 antigen. On the other hand,with the combined use of both α-lipoic acid and ambroxol as well asα-lipoic acid and enalapril, there was a detectable increase in theT-cell activation index in the normal range. This effect was observedwith early, intermediate and late activation markers. It can thus beconcluded that the normalization of the cellular thiol status achievedby the combined use of α-lipoic acid and the respective effectors of theglutathione metabolism is accompanied by a restitution of cell function.

EXAMPLE 3

[0059] Influence on the Cellular Thiol Status of Peritoneal Macrophagesin the Context of Renal Replacement Therapy

[0060] Peritoneal macrophages were isolated from the effluate ofperitoneal dialysis of patients with a high degree of renalinsufficiency, absorbed in a cell culture medium and incubated in agasifying incubation cabinet at 37° C., a relative humidity of 98% and7.5% relative air-CO₂ content. To investigate the influence of thecombinations used according to the invention on the thiol status of theperitoneal macrophages, individual fractions were treated with α-lipoicacid, the effectors of the glutathione metabolism ambroxol or the ACEinhibitor enalapril and with the combination of α-lipoic acid/ambroxolor α-lipoic acid/enalapril, while an additional fraction was maintainedas an untreated control.

[0061] The cellular thiol status was determined using the measurementmethods described in Example 1.

[0062]FIG. 4 illustrates the effect of the combination of α-lipoic acidand ambroxol (FIG. 4a) and α-lipoic acid and enalapril (FIG. 4b) in atime kinetic over 14 days (n−12).

[0063] When the individual substances were added, there was again anincrease of the cellular thiol expression only when α-lipoic acid wasused, while ambroxol and the ACE inhibitors had no effect. On the otherhand, with the combination of α-lipoic acid and ambroxol, beginningafter 72 hours, there was a significant increase of cellular thiolexpression, which achieved a super-additive effect after 4 days oftreatment and a maximum after 8 days, which exceeded the initial orcontrol data by a factor of three (FIG. 4a). The combination of α-lipoicacid and an ACE inhibitor (FIG. 4b) resulted in a similar, but onceagain significantly shortened time kinetic. In this case, a maximum ofthe super-additive effect was reached after only 48-72 hours oftreatment.

[0064]FIG. 5 shows the effect of the combination of α-lipoic acid andenalapril (FIG. 5b) on the membrane-resistant thiol expression ofperitoneal macrophages in the experimental system described above. Themembrane expression of thiols was determined on the basis of the medianfluorescence intensity (mfi) of the specimen (3000 cells/measurement)after coupling to a chlormethyl-fluorochrome derivative.

[0065] In comparison with the results of the intracellular thiolexpression, in this case there is a very clear effect of the addition ofα-lipoic acid by itself, although that effect disappeared after 4 daysof treatment. In contrast, the combined application of α-lipoic acid andambroxol (FIG. 5a) or α-lipoic acid and an ACE inhibitor (FIG. 5b)resulted in both a primarily more significant super-additive increase ofthe membrane-bound thiol expression, and one that was more stable overthe observation period.

EXAMPLE 4

[0066] Influence on the Phagocytosis Capacity of Peritoneal Macrophages

[0067] Phagocytosis capacity was selected as a measurement to make itpossible to characterize the peritoneal macrophages with regard to theiroriginal functions.

[0068] Peritoneal macrophages were isolated using a method analogous tothe method described in Example 3 and cultivated ex vivo.

[0069] The phagocytosis capacity was determined by a cytofluorimetrictest on the single-cell level. The macrophages were therebyco-cultivated with opsonized and fluorochrome-marked bacteria. Thenumber of bacteria absorbed during a defined period was determinedquantitatively by means of the fluorescence intensity in the macrophagesand was used as a measurement for their phagocytosis capacity.

[0070] The influence of the combinations used according to the inventionon the phagocytosis capacity of the peritoneal macrophages after atreatment time of 6 days is presented in the following table.Phagocytosis rate (mfi / 10,000 cells) Control 371 ± 39 α-lipoic acid[50 μM] 687 ± 59 Ambroxol [10 μM] 501 ± 52 α-lipoic acid + ambroxol1,398 ± 286  (p < 0.05) Enalapril [5 μM] 567 ± 59 α-lipoic acid +enalapril 1,698 ± 241  (p < 0.05) Captopril [10 μM] 653 ± 43 α-lipoicacid + captopril 1,589 ± 176  (p < 0.05)

[0071] After incubation with α-lipoic acid, ambroxol and enalapril, thephagocytosis rate was higher than that of the untreated control by afactor of 1.85 (α-lipoic acid), 1.35 (ambroxol) and 1.53 (enalapril). Onthe other hand, when the combination of α-lipoic acid and ambroxol wasused, there was an increase in the phagocytosis rate by a factor of 3.7.When the combination of α-lipoic acid and an ACE inhibitor was used, theincrease was by a factor of 4.6 (enalapril) and 4.3 (captopril).

[0072] Moreover, a direct correlation was established between thephagocytosis rate and the intracellular thiol content of the peritonealmacrophages for the combination of α-lipoic acid and ambroxol (r=0.79;p<0.01), α-lipoic acid and captopril (r=0.86; p<0.01), and α-lipoic acidand enalapril (r=0.82; p<0.01).

EXAMPLE 5

[0073] Influence on the Degree of Differentiation and Activation and theCytokine Synthesis of Peritoneal Macrophages

[0074] Peritoneal macrophages were isolated from patients during renalreplacement therapy using the method described in Example 3 andcultivated in the presence of the combinations of α-lipoic acid andeffectors of the glutathione metabolism according to the invention.After 6 days of incubation, the degree of differentiation of theperitoneal macrophages was determined by means of the expression of thecell surface antigens CD15 and CD11c, and the degree of cellularactivation was determined using cytofluorimetry by means of theco-expression of the activation antigens CD69 to CD15-positive cells andCD71 to CD11α-positive cells.

[0075] The results are presented in the following table: CD15 CD11cCD15/69 CD11c/71 Control 1.0 1.0 1.0 1.0 α-lipoic acid 1.18 ± 0.16 1.21± 0.11 1.09 ± 0.08 1.08 ± 0.09 [50 μM] Ambroxol 0.98 ± 0.13 1.01 ± 0.090.09 ± 0.11 0.96 ± 0.1  [10 μM] α-lipoic acid + 1.29 ± 0.21 1.65 ± 0.211.49 ± 0.13 1.83 ± 0.14 ambroxol Enalapril 1.21 ± 0.22 1.23 ± 0.22 1.19± 0.12 1.10 ± 0.14 [5 μM] α-lipoic acid + 2.12 ± 0.16 1.99 ± 0.15 1.69 ±0.2  1.58 ± 0.12 enalapril (p < 0.05) (p < 0.05) Captopril 1.19 ± 0.141.26 ± 0.24 1.69 ± 0.21 1.52 ± 0.16 [10 μM] α-lipoic acid + 2.25 ± 0.2 2.63 ± 0.23 1.74 ± 0.19 1.61 ± 0.18 captopril (p < 0.05) (p < 0.05)

[0076] It was shown that the expression of the maturation markers CD 15and CD11c increased markedly with the use of the combination of α-lipoicacid and ambroxol, and increased significantly with the use of thecombination of α-lipoic acid and ACE inhibitors. There was also asignificant increase in the activation antigens CD69 and CD71respectively in the respective cell populations. The application of thesingle substances had no effect or only a marginal effect on the degreeof differentiation and activation of peritoneal macrophages.

[0077] Simultaneously, in this experimental approach the cell cultureresidue was collected and the cytokines Interleukin-6 (IL-6) andInterleukin-1 receptor antagonist (IL-1ra) synthesized and secreted bythe peritoneal macrophages contained in the residue were determined. Theanalysis was performed using the enzyme immunoassay technique withstandardized measurement systems.

[0078] There was a significant reduction of the IL-6 synthesis in thepresence of the combination of α-lipoic acid and ambroxol and thecombination of α-lipoic acid and the different ACE inhibitors. Thiseffect in turn went significantly beyond the sum of the reductionachieved by the individual substances. The synthesis of IL-1ra underthese conditions was significantly induced. In this case, too, there wasa super-additive effect of the combination of α-lipoic acid and ambroxolor ACE inhibitors.

EXAMPLE 6

[0079] Influence on the Stability of Thiol Restitution in PeritonealMacrophages in the Dialysis Model

[0080] The combinations of thiol-restored peritoneal macrophages usedaccording to the invention were extracted from this test system after 6days and cultivated over a period of 14 days in a dialysis model. Forthis purpose the peritoneal macrophages were adapted to matrices thatwere coated with collagen IV and placed in contact with conventionalhigh-glucose dialysis solution 3 times a day for 60 minutes each time.In this case, the model was used for the induction of a combinedhyperglycemic/osmotic stress. FIG. 6 shows the effect of the combinationof α-lipoic acid and ambroxol (FIG. 6a) and α-lipoic acid and enalapril(FIG. 6b) on the intracellular thiol expression of the peritonealmacrophages in time kinetics. The membrane expression of thiol wasdetermined on the basis of the median fluorescence intensity (mfi) ofthe specimen (3000 cells/measurement) after coupling to achlormethyl-fuorochrome derivative. While with the primarilythiol-restored controls that had not been treated in this dialysismodel, there was a practically linear reduction of the intracellularthiol concentration within the first 4 days, the combined addition ofα-lipoic acid and ambroxol and of α-lipoic acid and enalapril resultedin a constant intracellular thiol status on the level of the primaryrestitution. Here, too, there is a mono-effect of α-lipoic acid,although it lasts only briefly and after approximately 4 days in thedialysis model is only approximately 50% as effective as thecombinations.

[0081] A similar result is apparent in the curves of the membrane-boundthiol expression illustrated in FIG. 7. Here again, the quantitiesobtained by the primary thiol restitution are kept constant by the useof the combination of α-lipoic acid and ambroxol (FIG. 7a) or ACEinhibitors (FIG. 7b), while with the addition of the individualsubstances, only intermediary (α-lipoic acid) or marginal effects(ambroxol, enalapril) were observed.

[0082] The effects of α-lipoic acid and effectors of the cellularglutathione metabolism on the cytokine synthesis of peritonealmacrophages after a treatment of 6 days (n=10) are presented in thefollowing table. IL-6 IL-1ra [ng/10⁶ cells] [ng/10⁶ cells] Control 53.1± 8.9 115.2 ± 23.4 α-lipoic acid [50 μM] 46.9 ± 6.7 119.8 ± 19.5Ambroxol [10 μM] 51.8 ± 8.1 118.6 ± 21.3 α-lipoic acid + ambroxol 31.5 ±9.2 126.8 ± 15.3 (p < 0.05) (p < 0.05) Enalapril [5 μM] 41.7 ± 7.3 121.1± 16.9 α-lipoic acid + enalapril 22.3 ± 8.8 139.8 ± 22.1 (p < 0.05) (p <0.05) Captopril [10 μM] 42.9 ± 7.7 129.4 ± 25.1 α-lipoic acid +captopril 28.1 ± 6.1 143.5 ± 18.7 (p < 0.05) (p < 0.05)

[0083] Overall, these tests make it plain that the application of thecombination of α-lipoic acid and the effectors of the glutathionemetabolism ambroxol and ACE inhibitors stabilizes a primarily massivelydamaged thiol status in different cell systems. As a result of thisnormalization, there is also a re-establishment of central cellularimmuno-regulatory functions, which is not achieved without such atreatment.

What is claimed is:
 1. Medicament containing an effector of theglutathione metabolism together with α-lipoic acid, its salts and/or itsprodrugs as a combination preparation for the simultaneous, separate ortemporally graduated treatment of a disruption of the thiol disulfidestatus in the treatment of diabetes mellitus.
 2. Medicament as recitedin claim 1, wherein the dose of the α-lipoic acid, its salts and/or itsprodrugs for application in human patients is between 30 and 1200 mg/d,preferably between 200 and 600 mg/d.
 3. Medicament as recited in one ofthe claims 1 or 2, wherein as the effector, ambroxol having the generalformula I

 its salts and/or its prodrugs is used.
 4. Medicament as recited inclaim 3, wherein the dose of Ambroxol, its salts and/or prodrugs usedfor application in human patients is between 7.5 and 90 mg/d, preferablybetween 60 and 75 mg/d.
 5. Medicament as recited in one of the claims 1or 2, wherein an Angiotensin Converting Enzyme (ACE) inhibitor is usedas the effector.
 6. Medicament as recited in claim 5, wherein the doseof the ACE inhibitor for application in human patients is between 0.2and 20 mg/d.
 7. Medicament as recited in one of the claims 1 to 6,wherein the ACE inhibitor used is1-[(2S)-3-mercapto-2-methylpropionyl]-L-prolin (captopril) having theformula II


8. Medicament as recited in one of the claims 1 to 7, wherein the ACEinhibitor used is1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-prolin(enalapril) having the formula III


9. Medicament as recited in one of the claims 1 to 8, wherein the ACEinhibitor used is (2S, 3aS,6aS)-1-{(S)-N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-alanyl}-octahydrocyclopenta[b]-pyrrol-2-carboxylicacid (Ramipril) having the formula IV


10. Medicament as recited in one of the claims 1 to 9, wherein themedicament is administered orally or parenterally.
 11. Medicament asrecited in one of the claims 1 to 10, wherein the medicament containsadditional additives selected from the group consisting of aqueoussolvents, stabilizers, suspension, dispersion and wetting agents. 12.Medicament as recited in one of the claims 1 to 11 in the form of asolution, a granulate, a powder, an emulsion, a tablet and/or a coatedtablet.
 13. Use of at least one effector of the glutathione metabolismtogether with α-lipoic acid, its salts and/or its prodrugs for themanufacture of a medicament for the treatment of a disruption of thethiol disulfide status in the treatment of diabetes mellitus.
 14. Use ofat least one effector of the glutathione metabolism together withα-lipoic acid, its salts and/or its prodrugs for the manufacture of amedicament in the form of a combination substance that modulatesimmunity and/or increases defenses in the context of the treatment ofdiabetes mellitus.
 15. Use of at least one effector of the glutathionemetabolism together with α-lipoic acid, its salts and/or its prodrugsfor the manufacture of a medicament that inhibits inflammation in thecontext of the treatment of diabetes mellitus.
 16. Use as recited in atleast one of the claims 13 to 15, wherein the effector of theglutathione metabolism and the α-lipoic acid, its salts and/or itsprodrugs are in a single formulation.
 17. Use as recited in at least oneof the claims 13 to 15, wherein the effector of the glutathionemetabolism and the α-lipoic acid, its salts and/or its prodrugs are inseparate formulations.